Sub-Minute Method for Determination of Naphazoline in the Presence of Diphenhydramine, Pheniramine or Chlorpheniramine by Capillary Electrophoresis

This paper presents a simple and low-cost capillary electrophoresis method for ultra-fast simultaneous determination of naphazoline (NPZ) and one of the following active ingredients: diphenhydramine (DIP), pheniramine (PHEN) or chlorpheniramine (CPHEN). The proposed method is based on capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-CD) equipped with a short length capillary column (10 cm). One sample can be analyzed every 35 s (ca. 100 injections per hour) with resolutions between peaks greater than 1.4. The optimized background electrolyte (BGE) was composed by 20 mmol L histidine and 10 mmol L 3,4-dimethoxycinnamic acid (pH = 9.5, adjusted with NaOH). Limits of detection were 25 μmol L for NPZ, DIP, and PHEN and 13 μmol L for CPHEN. The results obtained with the developed procedure were compared to those obtained by high-performance liquid chromatography (HPLC) and no statistically significant differences were observed (95% confidence level).


Introduction
Naphazoline (NPZ) is an active ingredient used in over-the-counter eye and nasal preparations. 1This drug is a long-lasting vasoconstrictor that is generally used as a topical nasal or ocular decongestant. 2,3Its field of action is increased by combining it with other active ingredients in the same formulation (synergistic effects), e.g.diphenhydramine hydrochloride (DIP), 4,5 pheniramine maleate (PHEN) 6 or chlorpheniramine maleate (CPHEN). 7,8PZ in combination with DIP is effective in relieving flu symptoms, such as nasal obstruction, runny nose and sneezing. 9The combination of NPZ with PHEN or CPHEN is commonly used as ophthalmic solution, due to the NPZ vasoconstrictor effect that improves the beneficial effects of PHEN or CPHEN (relief of redness, burning, irritation and dryness of the eyes). 10,11harmaceutical formulations containing NZP + DIP, NPZ + PHEN or NPZ + CPHEN are very popular and easily accessible, and therefore, are widely consumed around the world.For this reason, the development of fast, simple, low cost and environmentally friendly analytical methods for quality control of these drugs is desirable.Thus far, some analytical methods were reported for the simultaneous determination of NPZ and DIP, such as capillary electrophoresis (CE) with UV-Vis detection, 12 second-order derivative UV spectroscopy, 13,14 first-order derivative UV-spectroscopy, 15 and non-aqueous titration. 16or simultaneous determination of NPZ and CPHEN, two methods employing chemometric and derivative spectrophotometry were reported, 8,17 and for simultaneous determination of NPZ and PHEN, only one method based on high-performance liquid chromatography (HPLC) with UV-Vis detection was reported. 11However, most of the described methods require expensive instrumentation or is time-consuming, which is undesirable in quality control activities.
Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C 4 D) 18,19 is a technique based on the migration and separation of charged species under the effects of a high electric field. 20,213][24] The detection of charged species is based on the conductivity difference between the background electrolyte (BGE) solution and the charged analytes.][27][28][29] High-throughput methods are desirable especially for quality control routine analysis.Capillary electrophoresis fulfils this requirement due to the use of a short capillary column that provides fast and efficient analyte separation. 30,31n addition to the significant reduction in analysis time, an increase in sensitivity can also be achieved (shorter time for diffusion-concentration profiles).][34] In the present work, CE-C 4 D is explored to develop an ultra-fast method (around 35 s per analysis) for the simultaneous determination of NPZ and DIP, or NPZ and PHEN, or NPZ and CPHEN in three different samples.
Three pharmaceutical samples with different compositions were purchased from local drug stores: (i) 0.5 and 1.0 mg mL -1 of NPZ and DIP, respectively; (ii) 0.25 and 3.0 mg mL -1 of NPZ and PHEN, respectively; (iii) 0.5 and 0.5 mg mL -1 of NPZ and CPHEN, respectively.The excipients of nasal spray solution (i) are benzalkonium chloride, rose essence, and sodium chloride.The excipients of eye drops (ii and iii) consisted of boric acid, sodium borate, sodium chloride, disodium edetate and benzalkonium chloride.Samples were simply diluted in water before analysis.

CE measurements
Electrophoresis separations were performed in a homemade CE equipment with two compact and highresolution capacitively coupled contactless conductivity detectors (C 4 D). 18,35The detectors were positioned along the capillary at 10 cm from each end.The fused-silica capillary used in all experiments was 50 cm long (effective lengths of 10 and 40 cm) and 50 µm i.d.× 375 µm o.d.(Agilent, Folsom, CA, USA).The capillary was daily flushed with deionized water for 10 min before use, followed by 0.1 mol L -1 NaOH for 10 min, again with deionized water for 10 min and finally with BGE for 10 min.The samples were hydrodynamically injected for 1.25 s at 25 kPa.All experiments were carried out at +25 kV (inlet side) using normal electroosmotic flow (EOF) mode (towards the cathode).

Results and Discussion
In this study, the target compounds are weak bases (NPZ, pK a = 10.2;DIP, pK a = 8.9; both PHEN and CPHEN, pK a 1 = 3.6 and pK a 2 = 9.5) and, therefore, can exist in cationic forms in aqueous solutions. 37The chemical structures of the evaluated compounds are shown in Figure 1.
The initial intention was the determination of the four molecules in a single run, however, the similar mobilities of PHEN and CPHEN in the pH range (3 to 12) commonly used in capillary zone electrophoresis prevent their quick separation.However, all these active ingredients are not found in the same pharmaceutical formulation.The following mixtures are found in commercial products: (i) NPZ + DIP; (ii) NPZ + PHEN, and (iii) NPZ + CPHEN.Hence, efforts were made to identify a single BGE that allows the separation and detection of the target compounds in these samples.
The first BGE evaluated in this study was based on a previously optimized method for fast and simultaneous determination of NPZ and zinc (20 mmol L -1 of MES/HIS; pH = 6.0). 26Figure 2 shows the electropherograms obtained with the injection of two sample solutions suitably diluted in water (nasal spray solution and eye drops).
As can be observed in Figure 2, in addition to the active ingredients NPZ + PHEN (eye drops) and NPZ + DIP (nasal drops), both samples contain Na + as excipient.In the eye drop sample, an adequate resolution between the excipient (Na + ) and the analytes (NPZ and PHEN) was obtained.However, the concentration of Na + in the nasal spray sample is much higher and no adequate resolution between the peaks of Na + and the active ingredients (NPZ and DIP) was achieved.
In order to achieve better resolution between these target analytes and Na + , different BGE compositions were evaluated.In order to obtain BGEs with similar mobility (conductivity) to Na + , NaOH was added to weak acid solutions and the pH adjusted to values close to their respective pK a (maximum buffer capacity).Thus, the presence of Na + will no longer be detected by the C 4 D. Different BGEs composition commonly employed in capillary zone electrophoresis 38 were evaluated (Figure S1, Supplementary Information section): (S1a) 10 mmol L -1 boric acid with pH-adjusted to 9.5 with NaOH; (S1b) 20 mmol L -1 MES with pH-adjusted to 6.0 with NaOH; (S1c) 50 mmol L -1 CHES with pH-adjusted to 8.5 with NaOH; (S1d) 20 mmol L -1 CHES with pH-adjusted to 8.5 with NaOH; (S1e) 12 mmol L -1 TEA + 10 mmol L -1 DMX with pH-adjusted to 8.5 with NaOH; (S1f) 20 mmol L -1 HIS + 10 mmol L -1 DMX with pH-adjusted to 9.5 with NaOH.Best performance was achieved with the BGE composed by HIS and DMX and pH-adjusted to 9.5 with NaOH (Figure S1f).The electropherograms obtained with this buffer showed good resolution (r > 1.4) among the target analytes and nointerference of Na + (inactive excipient).The performance of the selected buffer can be better observed in Figure 3    The presence of high concentrations of Na + and benzalkonium chloride as excipients did not affect the resolution between the analyte peaks once similar electropherograms were observed in the absence (Figure 3a) and in the presence (Figure 3b) of the excipients.Finally, an electropherogram for a real nasal spray sample diluted in water (Figure 3c) was also carried out and the obtained result also leads to similar conclusion.
Some instrumental parameters such as separation voltage, injection time (at 25 kPa), temperature, and effective capillary length were also evaluated in order to obtain the optimal separation conditions.The best values were selected to achieve the best selectivity (resolution), good peak shape, sensitivity, minimal Joule effect (low currents), and shortest analysis time.Table 1 summarizes the evaluated ranges and the selected instrumental parameters of the proposed CE-C 4 D method.
The precision of the proposed method was evaluated by ten consecutive runs of standard solutions with similar composition of the three commercially available samples (Figure 4).The results obtained for each standard solution in the same day and with the same capillary column length were considered as intra-day precision.The results obtained for each standard solution in different days and with different column lengths (50 ± 3 cm) were considered as inter-day precision (Table 2).
Intra-day repeatability was below 3.5, 3.2 and 2.4% for NPZ and DIP, NPZ and PHEN, NPZ and CPHEN analysis, respectively.Inter-day repeatability was below 7.8% for all analytes.Linear response ranges were established considering correlation coefficients above 0.99 and the active ingredients concentration ratio in the samples.The results obtained in these studies and other analytical features are summarized in Table 2.

Table 1 .
Optimized conditions for the proposed CE-C4

Table 3 .
Results obtained by HPLC and by the proposed CE-C 4 D method CE: capillary electrophoresis; HPLC: high-performance liquid chromatography; E 1 : difference between results obtained by CE-C 4 D (capillary electrophoresis with capacitively coupled contactless conductivity detection) and the values reported in the label; E 2 : difference between results obtained by CE-C 4 D and by the reference procedures (HPLC); NPZ: naphazoline; DIP: diphenhydramine; PHEN: pheniramine; CPHEN: chlorpheniramine.